Pneumocystis jiroveci (Pneumocystis carinii) is an important cause of pneumonia (PCP) in immunocompromised individuals. The standard approach for diagnosing PCP is a microscopic examination of smears prepared from induced sputum or bronchial alveolar lavage (BAL) samples. Recently, investigators have been designing and testing PCR assays for the detection of P. jiroveci in respiratory samples. Of particular interest is the use of PCR with oral wash samples as a means of detecting P. jiroveci in the respiratory tract. These noninvasive specimens could prove to be of value for use in screening tests to rule out PCP. Microscopic methods are too insensitive to be useful with oral wash samples. The increased sensitivity of the PCR method, however, generates some positive results with samples obtained from patients who are only colonized or infected at a sub-clinical level. A precise quantitative method could help differentiate low level colonization from infection and, consequently, improve the clinical usefulness of PCR performed on oral washes and other respiratory samples. We have developed a rapid quantitative real time PCR assay targeting the MSG genes of P. jiroveci using fluorescence resonance energy transfer (FRET) detection probes for signal detection. A blinded, prospective study has been conducted with collaborators at UCSF to further evaluate the performance of the real time PCR assay in detecting PCP. For samples obtained within one day of the initiation of PCP therapy the sensitivity of oral wash samples with PCR for detecting PCP was greater than 90%. The results of this study are in print. A second collaborative study with UCSF is underway to attempt to detect P. jiroveci colonization of the upper airways of health care workers after exposure to patients with PCP. Recent work to improve the reproducibility of quantitative measurements of P. jiroveci in respiratory samples indicates that pre-treatment of sample materials with DTT greatly reduces the variability of serial quantitative P. jiroveci determinations. The blinded, prospective study of healthy Health Care Workers, who are not HIV-positive and did not have respiratory symptoms, was undertaken with 329 samples from 137 subjects using the DTT sample pre-treatment. A small percentage of these healthy individuals were repeatedly positive by the PCR assay and appear to be colonized with P. jiroveci. This data was presented at the Amercian Thoracic Society in May, 2005.